Bacteria of the genus Yersinia are responsible for a variety of human diseases. Y. pestis causes Bubonic Plague, and has recently regained prominence in public awareness due to its potential use as an agent of bioterrorism. In contrast, Y. pseudotuberculosis and Y. enterocolitica cause primarily gastrointestinal disease. However, despite the differences in disease symptoms, the three pathogenic Yersinia species are closely related, and share several common virulence determinants. Yersinia studies have provided fundamental insights into bacterial pathogenesis, including the first example of the widespread type III secretion system (TTSS). In Yersinia, as in all bacterial pathogens, many of the proteins that play important roles in virulence, including components of the TTSS, are located in the cell envelope. Under certain conditions, some envelope proteins become misfolded/mislocalized. Specific stress-response mechanisms deal with this problem, examples of which are the RpoE and Cpx systems of Escherichia coli and related organisms. These extracytoplasmic stress responses play important roles during host infection. The central hypothesis of this proposal is that a different extracytoplasmic stress response system is encoded by the phage-shock-protein locus (psp) of Y. enterocolitica. A Y. enterocolitica psp mutant is avirulent, and homologus psp loci are found in other bacterial pathogens, including Y. pestis and Vibrio cholerae. Our preliminary data indicate that the Psp system responds to mislocalization of several envelope proteins involved in virulence, including at least one component of a TTSS. By studying the Psp system we will gain further insight into the essential ability of bacteria to respond to stressful conditions that occur during host infection. Specifically, we propose to: (1) Analyze the proteins that induce the Psp system and characterize any overlap between Psp inducers and RpoE/Cpx inducers; (2) Determine the topology of the Psp system, and investigate how Psp allows extracytoplasmic stress to be sensed and signaled across the cytoplasmic membrane; (3) Characterize genes directly controlled by the Psp system, in order to identify further stress response components.